Hydrogen sulfide (H2S) is a newly identified modulator. It is synthesized in mammalian tissues from cysteine and homocysteine by the enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE).
Hydrogen sulfide (H2S) protects the gastric mucosa from injury, reduces inflammation of mucous membranes and enhances tissue repair. Dysfunction of hydrogen sulfide (H2S) homeostasis is associated with ulcerative colitis, hypertension, atherosclerosis, cardiomyopathy, diabetic vascular disorders and diabetic nephropathy.
Current methods for detecting hydrogen sulfide (H2S) in living cells or tissues include gas chromatographic, electrochemical and colorimetric methods. However, these methods require processing and/or breakdown of tissue and cell lysates. At present, no method capable of detecting hydrogen sulfide (H2S) without damaging cells or tissues is available.
Although fluorescence imaging using a one-photon fluorescent dye is known as a method for detecting cells nondestructively, the one-photon fluorescent dye has to be excited with monochromatic light (˜350-550 nm). Accordingly, there arise problems such as low penetration depth, photobleaching and cellular autofluorescence. In order to overcome these problems, two-photon microscopy (TPM) uses a two-photon probe capable of detecting the target substance deep in the living cells and tissues.
Two-photon microscopy is a new technique that allows imaging of living tissue up to a very high depth for a long period of time using two photons of low energy (Helmchen, F.; Denk, W. Nat. Methods 2005, 2, 932; Zipfel, W. R.; Williams, R. M.; Webb, W. W. Nat. Biotechnol. 2003, 2, 1369; Kim, H. M.; Cho, B. R. Acc. Chem. Res. 2009, 42, 863; Kim, H. M.; Cho, B. R. Chem. Asian J. 2011, 6, 58).
Korean Patent Publication No. 2010-0000044 discloses a two-photon dye for imaging acidic vesicles in vivo and a method for imaging acidic vesicles using the same. The two-photon dye selectively binds to acidic vesicles present in the cytoplasm and emits two-photon excited fluorescence.
Although a method for imaging acidic vesicles in vivo using a two-photon fluorescent dye has been presented as described above, there is no report about detection of hydrogen sulfide (H2S).
Accordingly, development of a ratiometric two-photon fluorescent dye capable of detecting hydrosulfide ion and total sulfide in vivo without damaging cells or tissues is needed.